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From The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, and Department of Cell and Neurobiology, The Keck School of Medicine of the University of Southern California, Los Angeles, California.
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Abstract |
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METHODS. Weri-Rb-1 retinoblastoma cells were cultured in the absence or presence of RA for various periods. DNA microarray analysis profiled gene expression followed by real-time PCR and Northern and immunoblot analyses to confirm the change in expression of selected retinal genes and their gene products. Additional methodology included flow cytometry analysis, immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.
RESULTS. DNA microarray analysis of approximately 6800 genes revealed RA-induced upregulation of cone-specific genes and downregulation of rod-specific genes in Weri-Rb-1 cells. Other significantly upregulated mRNAs included chicken ovalbumin upstream promoter-transcription factor (COUP-TF1), retinoid X receptor (RXR)-
, thyroid hormone receptor (TR)-ß2, and guanylyl cyclase-activating protein (GCAP)-1. Real-time PCR and/or Northern blot analysis confirmed the expression changes of a subset of genes including the upregulation of a pineal- and retina-specific transcription factor, CRX. RA treatment also led to G0/G1 cell cycle arrest and increased both the intensity of human cone arrestin (hCAR)-immunoreactivity and the number of apoptotic cells. The cell-cycle–arrest stage correlated with the observed microarray results in which the RA treatment downregulated critical genes such as cyclins (cyclin E, cyclin D3) and cyclin-dependent kinases (CDK5, CDK10).
CONCLUSIONS. These data suggest that RA induces a subpopulation of retinoblastoma cells to differentiate toward a cone cell lineage while selectively leading other cells into apoptosis.
With the advent of DNA microarray technology, the simultaneous study of global gene expression patterns of human retinal genes is feasible. Also, these new advances provide an efficient means of gaining critical insights into the expression, regulation, and potential function of genes that may contribute to human photoreceptor development, for which information is not currently available. To this end, we used commercially prepared microarrays (huGene FL; Affymetrix, Santa Clara, CA) to examine the expression of 6800 known genes in Weri-Rb-1 retinoblastoma cells and their RA-induced, differentiated progeny. These gene expression profiles are highly reproducible and represent modifications of a variety of molecular markers, including genes involved in phototransduction pathways; those expressing transcription factors, growth factors and their receptors, and cytokines and their receptors; and those serving as intracellular signal-transduction modulators and effectors. Our investigation was focused on the rod and cone photoreceptor-specific genes and the evaluation of the parameters of cell cycle, differentiation, and apoptosis of Weri-Rb-1 cells treated with RA. Moreover, our attention was also concentrated on the potential role of retina-specific transcription factors in the developmental cell fate when treated with RA.
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Materials and Methods |
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Microarray Probe Preparation, Hybridization, and Scan
The RNA samples were processed as recommended by the manufacturer (Affymetrix). Briefly, total RNA was isolated from Weri-Rb-1 cells treated with either 10 µM of ATRA or DMSO (control) for 3 hours, 48 hours, or 7 days, using extraction reagent (Trizol; Life Technologies, Inc., Gaithersburg, MD). Reverse transcription was performed on 10 µg total RNA with the use of a kit and an oligo(dT)24-anchored T7 primer (SuperScript Choice System; Life Technologies, Inc.). After second-strand synthesis, the double-stranded cDNA was cleaned up by extraction with phenol-chloroform-isoamyl alcohol and recovered by ethanol precipitation. A transcript labeling kit (BioArray HighYield RNA Transcript Labeling Kit; Enzo Diagnostics, Farmingdale, NY) was used for the production of hybridizable biotin-labeled RNA targets by in vitro transcription from T7 RNA polymerase promoters. The cDNA prepared from total RNA was used as a template in the presence of a mixture of unlabeled ATP, CTP, GTP, and UTP and biotinylated CTP and UTP. In vitro transcription products were purified (RNeasy Mini Kit; Qiagen, Valencia, CA) to remove unincorporated NTPs and fragmented by incubation at 94°C for 35 minutes. The fragmented sample cRNA (complementary RNA) was stored at -20°C until the hybridization was performed.
The following steps were then performed at the Children’s Hospital LA Microarray Core Facilities. Gene microarray chips (huGene FL GeneChips; Affymetrix) containing 6800 genes were used to profile mRNA expression. The biotinylated cRNA (10 µg/chip) was hybridized for 16 hours at 40°C to a set of oligonucleotide arrays in a hybridization system (GeneChip Fluidics Station 400; Affymetrix). After hybridization, the microarray underwent a series of stringency washes and was stained with streptavidin-conjugated phycoerythrin. Probe arrays were scanned with a confocal laser scanner (Affymetrix).
Data Analysis
Hybridization data from text files were imported to a spreadsheet (Excel; Microsoft, Redmond, WA). Data analysis was performed with software developed by the microarray manufacturer (Genechip Analysis; Affymetrix) and the multiples of change between the hybridization intensities of RA-treated and control samples were obtained.
Real-Time PCR
Three micrograms of total RNA from Weri-Rb-1 cells treated with either 10 µM of ATRA or DMSO (control) for 48 hours or 7 days were reverse transcribed with 200 U reverse transcriptase (Superscript II RNase H-; Invitrogen, San Diego, CA) in a volume of 20 µL, with 100 µM random primers (Invitrogen) used according to the manufacturers’ instructions. The quantification of the selected 15 genes by real-time PCR was performed on a fluorescence detection system (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany). Oligonucleotide primers were designed using the accompanying software (LightCycler Probe Design Software; Roche Molecular Biochemicals). The nucleotide sequences of the primers used are shown in Table 1 . The optimal PCR reactions for all investigated genes were established with a kit (LightCycler Fast Start DNA Master SYBR Green I Kit; Roche Molecular Biochemicals), according to the manufacturer’s instructions. Annealing temperatures and MgCl2 concentrations were optimized to create a one-peak melting curve. In addition, the PCR reactions were recovered after each PCR analysis and amplicons were checked by agarose gel electrophoresis for a single band of the expected size.
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The reaction product was characterized by the point during cycling when amplification of PCR products was first detected (crossing point) with the manufacturer-provided software (LightCycler, ver. 3.5; Roche Molecular Biochemicals). The results are presented as the ratios of mRNA expression in treated cells in relation to the amount present in untreated cells and are normalized to an internal control gene, ß-actin, for different mRNA input and reverse transcript efficiencies.
Northern Blot Analysis
Northern blot analysis was performed as previously described.1 2 Probes used for hybridizations were prepared by RT-PCR with RA-treated or untreated Weri-Rb-1 cell cDNA as a template and verified by DNA sequencing.
Immunoblot Analysis
Immunoblot analysis was performed as described.2 The immobilized proteins were detected on the membrane with an enhanced chemiluminescence kit with affinity-purified anti-bovine CRX peptide polyclonal antibody3 or anti-human thyroid hormone receptor (TR)-ß2 (N-16) peptide goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1: 1000 and 1:100 dilutions, respectively. Anti-rabbit (Bio-Rad Laboratories, Richmond, CA) or anti-goat (Santa Cruz Biotechnology) secondary antibodies were used at 1:10,000 and 1:5,000 dilutions.
Determination of Cell Cycle Stage by Fluorescence-Activated Cell Sorting
Cellular DNA content was analyzed with a flow cytometry system (FACScan; BD Bioscience Immunocytometry Systems, San Jose, CA) at the Norris Cancer Center Core Flow Cytometry Facility of the Keck School of Medicine of the University of Southern California (USC). Briefly, treated or untreated Weri-Rb-1 cells were harvested and washed, fixed in cool 70% ethanol for at least 2 hours, and then incubated with 1 mL propidium iodide/Triton X-100 staining solution with RNase A for either 15 minutes at 37°C or 30 minutes at room temperature. For each cell population at least 10,000 cells was analyzed by flow cytometry. The proportion in G0/G1, S, and G2/M phases was estimated by using the cell cycle analysis software (Cell Quest; BD Bioscience).
Immunocytochemistry
Weri-Rb-1 cells from suspension culture were gently dissociated and plated onto poly-D-lysine eight-well chamber slides (Cellware; BD Bioscience) at 3 x 105 cells/mL and maintained for 24 hours before the addition of RA (10 µM) or DMSO. Medium was changed and chemicals were reapplied every 2 days. After treatment for 5 days, medium was removed, and the cells were processed for immunofluorescent staining with the affinity-purified anti-hCAR polyclonal peptide antibody LUMIF,1 and a Cy3-conjugated goat anti-rabbit secondary antibody, as described.3 The slides were mounted and photographed with a confocal microscope (Carl Zeiss, Inc., Oberkochen, Germany).3
Analysis of Apoptosis
To detect apoptosis in individual cells, Weri-Rb-1 cells were seeded onto poly-D-lysine eight-well chamber slides and treated as described earlier in the immunocytochemistry study. After a 48-hour treatment with either RA or DMSO, cells were fixed in 4% paraformaldehyde in PBS for 25 minutes at 4°C, rinsed with PBS and permeabilized for 5 minutes on ice in 0.2% Triton X-100 in PBS. Apoptotic cells were visualized by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay,4 with an apoptosis detection system (Apoptosis Detection System; Promega), according to the manufacturer’s instructions. After labeling with fluorescein-12-dUTP, the slides were rinsed with 2x SSC, and propidium iodide (PI) was added to stain all cells. The slides were then mounted and photographed. Apoptotic cells were quantified by counting cells labeled with fluorescein-12-dUTP in 10 random fields per condition under a microscope. The total number of apoptotic cells was divided by the total number of cells stained with propidium iodide in the same field, yielding the percentage of apoptotic cells within each specified field.
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Results |
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subunit, green cone pigment gene, and cone transducin alpha subunit, after either 48 hours or 7 days of treatment. Guanylyl cyclase-activating protein (GCAP)-1, which has a higher expression level in cones than in rods,8 increased by 2.2-fold after 3 hours of RA treatment and continued to increase with longer treatment. TRß2, which is essential in the development of green cone photoreceptors,9 increased by 7.2-fold after 48 hours, but decreased 5-fold after 7 days of RA treatment. The retinoic acid receptor (RXR)- was upregulated after both 48 hours (3.3-fold) and 7 days (2.8-fold) of RA treatment. Expression of chicken ovalbumin upstream promoter-transcription factors (COUP-TF1) increased 4.4-, 16.2-, and 13.6-fold after 3 hours, 48 hours, and 7 days of RA treatment, respectively.
A dramatic contrast was observed in visual genes associated with components of the rod phototransduction pathway. All three subunits of rod transducin (, ß, and ) and the rod photoreceptor cGMP-gated channel were each downregulated significantly, as shown in Table 3 . Cone rhodopsin-sensitive cGMP 3'5'-cyclic phosphodiesterase gamma subunit (PDE6H), which is expressed in blue cone photoreceptor cells, was also among those downregulated genes. Adenylyl cyclase type II was downregulated dramatically after both 48 hours (55.1-fold) and 7 days (71.5-fold) of RA treatment. It is notable that transducin-like enhancer protein-1, a human protein homologous to Drosophila groucho (Gro) that affects neuronal fate through negative regulation of gene expression,10 11 decreased 2.2-, 30-, and 4.9-fold after a 3 hours, 48 hours, and 7 days of RA treatment, respectively. Major histocompatibility complex (MHC) class I, previously identified in differentiated Y79 cells after RA treatment12 and essential in central nervous system development,13 increased 2.9-fold after 3 hours and 6.2-fold after 48 hours.
Another prominent observation of our study is that numerous cell cycle regulatory proteins, including cyclins, cyclin-dependent kinases (cdks), cdk inhibitors (CKIs), and the E2F families of proteins, which constitute a network of interacting factors that govern exit from or passage through the mammalian cell cycle, were downregulated by RA treatment, whereas the retinoblastoma (Rb) family members Rb1 and Rb-related protein p107 were upregulated after 48 hours of RA treatment. In contrast, the DNA fragmentation factor-45 -subunit and caspase 8, which are coupled with apoptosis,14 15 were increased 17.6-fold and 3.2-fold, respectively, after 48 hours.
Rb is a tumor suppressor involved in the transcriptional regulation of genes required for the G1-to-S phase transition of the cell cycle,16 and the mutation of this gene is the cause of hereditary retinoblastomas.17 Whereas all normal tissues and many tumor cells express an Rb mRNA of 4.7 kb, retinoblastomas were found either to have no Rb expression or to have Rb transcripts of reduced size (4.0 kb).17 Weri-Rb-1 has a homozygous deletion of the entire Rb gene, whereas Y79 contains a partial deletion of one allele and an uncharacterized mutation in the other.18 19 20 In our experimental system, the upregulated Rb mRNA in Weri-Rb-1 cells by RA treatment is most likely a cross reaction with other Rb-related genes that are upregulated by RA.
Validation of Array Results by Real-Time PCR
Eleven genes with significant expression changes by microarray analysis, including those expressed in either cone or rod photoreceptors, were selected for confirmation of their relative expression by real-time quantitative PCR. Because of the importance of photoreceptor-specific transcription factors in the regulation of rod and cone photoreceptor differentiation, we also determined the relative change in expression of CRX, a homeobox gene3 21 22 ; OTX2, another member of the homeodomain-containing transcription factors, which is highly homologous to CRX and expressed in brain and retina23 24 25 ; neuroretinal leucine zipper (NRL)26 27 28 29 ; and NR2E3, a photoreceptor-specific nuclear receptor.30 31 32 The quality of the real-time PCR products is shown in Figure 1 , and the relative multiples of change in expression of these genes as determined by microarray, real-time PCR, and Northern blot analyses are presented in Table 4 .
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Confirmation of CRX and TRß2 Upregulation by RA
CRX is a homeobox gene specifically expressed in the photoreceptors of the developing and mature retina and is crucial in rod and cone photoreceptor differentiation.21 22 Previously, we identified potential CRX elements in the cone arrestin promoter.2 34 Because the microarrays (huGene FL; Affymetrix) did not contain the CRX gene, Northern blot analysis was used to examine CRX gene regulation by RA in Weri-Rb-1 and Y79 cells. A CRX-specific cDNA fragment, which is outside the homeodomain, was radioactively labeled and used as a probe for Northern blot analysis. As shown in Figure 2A , the expression of both the 3.9- and the 2.5-kb mRNA of CRX was induced dramatically by 6-day RA treatment in both cell types. To analyze the dynamics of this induction, a dose–response and time-course analysis of CRX mRNA expression was analyzed after RA treatment of Weri-Rb-1 cells. Figure 2B shows that RA enhanced CRX mRNA levels in a dose-dependent manner and that the maximal effect was achieved with 3 µM RA. In the time-course experiments, the expression of CRX mRNA was elevated above basal levels after 4 days of exposure to RA (Fig. 2C) . Immunoblot analysis confirmed that the immunoreactive CRX protein was upregulated after 6 days of RA treatment (Fig. 2D) . Immunoreactive NRL, a rod-specific transcription factor, was below detectable levels in these cells, whether treated or untreated with RA, by immunoblot analysis (antisera generously provided by Anand Swaroop, Department of Ophthalmology & Visual Sciences and Eccles Institute of Human Genetics, University of Michigan, Ann Arbor, MI; data not shown). These data suggest that CRX may have a role in a later stage of retinoblastoma cell differentiation due to RA treatment.
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Discussion |
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In prior work, we examined the molecular mechanisms involving both the mRNA transcription and the protein translation of cone arrestin expression that are dramatically increased with RA treatment of retinoblastoma cells.2 Because RA induces hCAR expression in retinoblastoma cells through the upregulation of RXR, which is mainly localized to the cone photoreceptors in the human retina, it would be interesting to know whether RA induction of hCAR is accompanied by transcription alterations of other cone-specific genes. With oligonucleotide array-based expression-profiling technology (Affymetrix), we observed that the expression pattern of a broad network of genes was modulated in a time-dependent manner by RA treatment. In addition, with RA treatment, the retinoblastoma cell line underwent G0/G1 cell cycle arrest and displayed conelike genetic differentiation and selective apoptosis.
Cell differentiation is a coordinated process that includes cell cycle exit and tissue-specific gene expression.48 The present study is of particular interest and significance because RA upregulated a subset of cone-specific genes during the differentiation of retinoblastoma cells. These results are consistent with the established concept that RA levels influence cell fate and mediate the differentiation of specific neuronal phenotypes during retinal histogenesis.38 49 In cultured embryonic rat or fetal human retinas, RA promotes retinal progenitor cells to develop into rod photoreceptors.39 Exogenous RA also promotes rod photoreceptor differentiation in rat retina in vivo when injected in pregnant rats at days 18 and 20 of gestation.39 Moreover, application of RA to zebrafish embryos causes precocious rod photoreceptor development and inhibition of the maturation of cone photoreceptors.50 The results presented in this article are in conflict with the published observations, in that RA induced retinoblastoma cells to differentiate into cone photoreceptors, but it inhibited rod gene expression. This discrepancy could be explained in at least three ways. First, the effect of RA on cell differentiation during embryonic development is probably tightly controlled by timing. All the published results have been obtained with embryonic retinas, but the retinoblastoma cell lines were developed from juvenile tumor tissues, which had passed the embryonic developmental stage. Indeed, a more recent observation reveals that RA induces rod photoreceptor-selective apoptosis during postnatal development of mouse retina.51 Second, the retinoblastoma cells used in our study may differ from developing retinal cells in that certain diffusible induction factors normally found in the microenvironment of the embryo may be either missing or present in overabundance. A third alternative is the different cell–cell interaction involved in the RA-induced cell differentiation between normal retinal development and retinoblastoma cell differentiation. In normal retinal development, photoreceptors arise from a population of precursor cells that are multipotent and intrinsic in the retina. The first photoreceptors to form are the cones, with the rods appearing much later.52 However, in retinoblastoma, neoplastic photoreceptors, mostly red/green–sensitive cones, and glia cells (Müller cells) constitute the overwhelming majority of the cells.53 It seems likely that RA induces retinoblastoma cells along a red/green-cone pathway due to a red/green-cone–dominant microenvironment.
We have also shown that RA downregulates rod-specific genes during retinoblastoma cell differentiation. Given the example that RA induces rod-selective apoptosis during postnatal retinal development,51 it easily led us to postulate that RA also induces the rodlike cell population in the heterogeneous retinoblastoma cell line to choose a cell death pathway. This assumption is supported by the increased expression of a DNA fragmentation factor, caspase 8, and the increased number of apoptotic cells in RA–treated Weri-Rb-1 cells compared with the DMSO-treated control.
Microarray analysis also revealed that RA treatment of Weri-Rb-1 cells resulted in an induction of expression of genes in the steroid-thyroid receptor superfamily, such as RXR, TRß2, and COUP-TF1, plus a photoreceptor-specific homeobox gene, CRX. Previously, we identified that both Weri-Rb-1 and Y79 cells express all subtypes of RAR and RXR transcripts, and RXR is potentially the RA receptor subtype involved in human cone arrestin regulation by RA.2
TRß2 is a member of the steroid-thyroid receptor superfamily that plays a key role in cone-specific gene expression. Thyroid hormone receptors are located in the developing chicken retina54 55 with the TRß2 isoform being expressed in cones.54 Northern blot analysis revealed a prominent expression of TRß2 mRNA in mouse eye development, which peaked at approximately embryonic day 17.5 and declined in the postnatal period, similar to the pattern in chick.9 In recent mouse gene-knockout studies, deleting the THRB gene (encoding TRß2) caused the selective loss of green (M) cones and a concomitant increase in S-opsin immunoreactive cones.9 Our microarray and Northern blot data are consistent with this study, because the upregulation of TRß2 is followed by the increase in expression of green cone pigment gene after RA treatment. Furthermore, the human cone arrestin promoter also contains an element (DR-4) that strongly resembles an authentic thyroid hormone response element (TRE).2 These results suggest that TRß2 may play an important role in driving cone-specific gene expression, both in developing retina and in retinoblastoma differentiation.
Several studies have suggested that COUP-TF may be a part of the retinoid signaling pathway, both in vivo and in cell culture systems.56 57 58 Upregulation of COUP-TFI and COUP-TFII genes also occurs in differentiation programs of P19 embryonic carcinoma (EC) cells triggered by retinoic acid (RA).59 The biological importance of COUP-TF in retina development was demonstrated with the Drosophila homologue seven-up (SVP). SVP is essential to specify photoreceptor subtype in the development of the compound eye.60 61 Lu et al.62 further demonstrated that in the mouse, bovine, or human rod arrestin gene promoter, a DR-7 element (TGACCT of direct repeat with a 7-bp spacer), mediates the positive transcriptional effect of COUP-TF. The mouse retinal expression pattern of COUP-TF in embryonic day 14 coincided with the initial expression of the rod arrestin gene. No COUP-TF transcripts were detected in either adult retina or Weri-Rb-1 cells,62 leading them to propose that COUP-TF may effectively regulate only the expression of the rod arrestin gene in the developing retina, whereas other factors regulate this gene in the adult retina and in retinoblastoma cells.62 We demonstrated for the first time that RA rapidly induces expression of COUP-TF1 in Weri-Rb-1 cells, and the expression of COUP-TF1 is associated with retinoblastoma cell differentiation, implying that COUP-TF1 plays a crucial role, not only in the control and timing of rod-specific programs during retinal development, but also in cone-specific programs during the differentiation of cone photoreceptors.
CRX is the first major photoreceptor-specific homeodomain transcription factor to be identified.21 22 63 Although CRX is unlikely to be the dominant transcription factor involved in expression of the cone arrestin and cone transducin alpha subunit (GNAT2) genes, despite the presence of three CRX binding sites in the cone arrestin promoter,2 we could not exclude the green cone pigment gene from transactivation by CRX, because it was expressed only after 7 days of RA treatment in Weri-Rb-1 and was absent in CRX-/- mice.64 The requirement for CRX in transcription of the cone opsin loci in mice supports the notion that the human red/green locus requires CRX activity at the CRX-binding element in the locus control region.22
In summary, our findings imply that RA mediates induction that coordinates cone-specific gene activation, rod-specific gene inactivation, and cell cycle arrest during the differentiation of retinoblastoma cells. The molecular events involved depend on the modulation of two main opposing switches that contain members of the steroid-thyroid receptor superfamily and cell cycle regulators. This may represent a general mechanism by which retinoids signal cell cycle control and cell fate biases that may have therapeutic implications in the pharmacologic triggering of growth suppression and destruction of tumor cells. Even more important, the fact that RA induces most of these cells to differentiate into a more conelike phenotype may someday be part of a viable option for therapeutic rescue of the cone photoreceptors before their destruction leads to blindness.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 2, 2002; revised October 29, 2002; accepted November 13, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Cheryl M. Craft, Chair, Department of Cell and Neurobiology, The Keck School of Medicine of the University of Southern California, 1333 San Pablo Street, BMT 401, Los Angeles, CA 90089-9112; ccraft{at}usc.edu.
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